CN111308103B - Cardiopulmonary quintuplet detection kit, rare earth nano fluorescence detection card and detection method thereof - Google Patents
Cardiopulmonary quintuplet detection kit, rare earth nano fluorescence detection card and detection method thereof Download PDFInfo
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- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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Abstract
The invention discloses a heart-lung quintuplet detection kit, a rare earth nanometer fluorescence detection card and a detection method thereof, which can accurately and quantitatively detect the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in human blood, can avoid the fluorescence interference of a sample by utilizing a rare earth nanometer fluorescence microsphere detection technology, and the fluorescence microsphere is a rare earth fluoride near-infrared two-region luminescent nano material and has the advantages of low background, long luminescent life, strong fluorescence signal, high signal to noise ratio and the like.
Description
Technical Field
The invention relates to a heart-lung quintuplet detection kit, a rare earth nanometer fluorescence detection card and a detection method thereof.
Background
Troponin (Troponin) is composed of a trione of Troponin I, T, C, which together with tropomyosin regulates actin and myosin interactions by regulating the activity of Ca2+ on striated actin atpase. After myocardial injury, the cardiac troponin complex is released into the blood and after 4-6 hours, begins to rise in the blood, and the elevated troponin I can remain in the blood for 6-10 days, providing a longer detection period. The cardiac troponin I (cTnI) has high cardiac muscle specificity and sensitivity, so the cardiac troponin I (cTnI) becomes an ideal cardiac infarction marker at present.
Myoglobin (Myoglobin, Myo) is a binding protein consisting of a peptide chain and a heme prosthetic group, a protein that stores oxygen in muscle. The rise can occur within 2 hours as soon as the onset of chest pain occurs; severe congestive heart failure and cardiac surgery patients also have increased due to the presence of myocardial damage. Myoglobin is a sensitive index for diagnosing acute myocardial infarction, so that myoglobin becomes one of current myocardial infarction markers.
Creatine Kinase (CK) has four isoenzyme forms: muscle type (MM), brain type (BB), hybrid type (MB), and mitochondrial type (MiMi), with MB type being predominantly present in cardiomyocytes. During myocardial infarction, creatine kinase is increased within 6 hours of attack, the peak is reached within 24 hours, and the creatine kinase is recovered to be normal within 3-4 days, wherein creatine kinase isoenzyme CK-MB has higher diagnosis specificity, so that creatine kinase isoenzyme CK-MB becomes one of the current myocardial infarction markers.
D-Dimer (D-Dimer) is a specific degradation product of crosslinked fibrin after plasmin hydrolysis, and has important significance in diagnosing and treating fibrinolytic system diseases (DIC, various thrombi) and diseases related to the fibrinolytic system, and monitoring thrombolytic therapy. The water content of fibrin degradation product D is increased, indicating that frequent fibrin degradation processes exist in the body. Thus, fiber D dimers are key indicators of Deep Vein Thrombosis (DVT), Pulmonary Embolism (PE), Disseminated Intravascular Coagulation (DIC).
BNP belongs to natriuretic peptide family, is a peptide hormone, has effects of promoting urination, promoting natriuresis, resisting renin-angiotensin-aldosterone system, dilating blood vessel and lowering blood pressure, and is involved in regulating water-salt balance of human body. NT-proBNP is a biologically inactive amino acid fragment produced during the formation of BNP. The normal human body has extremely low NT-proBNP level, and BNP secretion is stimulated when ventricular pressure is changed and the wall tension is increased due to volume load, so that the increase of BNP/NT-proBNP concentration in blood can well reflect the change of ventricular structure and function, and the heart has no damage or injury degree. A large number of basic and clinical researches show that BNP/NT-proBNP is the most sensitive and specific index of heart dysfunction, is an objective marker with epoch-making significance for diagnosing heart failure, can more truly reflect the change of heart function compared with the heart function classification of the American New York Heart Association (NYHA), and is beneficial to finding heart failure at an early stage or a pathological slight stage and distinguishing asymptomatic or slightly symptomatic heart failure patients (NYHA grade and II grade) from non-heart failure patients. Has good guiding significance for risk stratification, prognosis evaluation, monitoring of the treatment effect of the heart failure and the like of patients with chronic heart failure
cTnI, CK-MB and Myo are good high-specificity indexes for diagnosing Acute Myocardial Infarction (AMI) at an early stage, and NT-proBNP and cTnI are effective indexes for diagnosing Acute Coronary Syndrome (ACS) and have a certain correlation with the lesion severity of ACS. The cTnI and the NT-proBNP have higher specificity for diagnosing Chronic Heart Failure (CHF), and meanwhile, the NT-proBNP, the cTnI and the D-Dimer have higher clinical diagnosis significance for diagnosing Pulmonary Embolism (PE), so that the combined detection of the five indexes is more helpful to early and accurately diagnosing the heart and lung functions, and has clinical guiding significance.
In the prior art, the cardiac marker is only detected aiming at a single index, namely three cardiac muscle cTnI, CK-MB and Myo indexes, the five heart-lung joint detection is less, and in addition, the detection method mainly comprises an immunoturbidimetry method, a fluorescence immunochromatography method, a colloidal gold method and the like. The colloidal gold method has the advantages of simple and quick operation, but low sensitivity and inaccurate quantification; the immunoturbidimetry is sensitive and accurate, can be applied to a full-automatic biochemical analyzer, but needs an instrument and consumes a long time, is suitable for processing a large number of samples, and cannot meet the aim of rapid detection; the immunofluorescence method is to covalently bond the cTnI/CKMB/Myo/D-Dimer/NT-proBNP antibody on a surface active group of a fluorescent microsphere, judge the result by whether the detection line generates fluorescence after excitation, is rapid, convenient and accurate to quantify, has the advantages of high sensitivity, stable marker and the like, and is widely applied to the field of medical immunodetection.
However, many compounds and proteins in biological fluid and serum can fluoresce, the spectrum range of the commonly used fluorescent marker is mostly in a visible region (400-750 nm), and the fluorescent marker using the visible region can generate strong background fluorescence, interfere detection signals and reduce the detection sensitivity. And the emission wavelength range of the near-infrared fluorescent material is 650-1000 nm, and in this range, the organism autofluorescence is weaker, so that the use of the near-infrared luminescent material to mark the antibody can make the fluorescent test strip have low background fluorescence, the detection signal-to-noise ratio is high, and the detection sensitivity is improved.
In addition, the rare earth nano material is a widely used biomarker, and compared with the traditional marker, the rare earth doped nano material has the advantages of stable physical and chemical properties, narrow-band emission, long service life and the like, so that excitation light interference is eliminated, background fluorescence interference can be eliminated through time-resolved delay detection, and the biomarker is not easily influenced by the environment in the process of biomarker. The rare earth luminescent material, especially the rare earth fluoride, has low phonon energy and stable physical and chemical properties, is suitable for the matrix material of the rare earth luminescent material, and can reduce the non-radiative relaxation of the excited state of the active ion so as to improve the luminous efficiency of the active ion. Therefore, the rare earth fluoride near-infrared two-zone luminescent nano particles are used as markers, the luminescent service life is long, the emission wavelength is 808nm, the background fluorescence of the test strip can be reduced, the time-resolved fluoroimmunoassay technology is combined, two parameters of the detection wavelength and the detection time are used for signal resolution, the interference of non-specific fluorescence can be effectively eliminated, and the analysis sensitivity and the accuracy are greatly improved.
Therefore, the development of the rare earth nano fluorescence detection card which has wide detection range, high sensitivity, high detection accuracy and capability of rapidly determining the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in blood, and the cardiopulmonary quintuplet detection kit and the detection method using the detection card have important significance.
Disclosure of Invention
The invention aims to provide a rare earth nano fluorescence detection card, a heart-lung quintuplet detection kit adopting the detection card and a detection method aiming at the defects of the prior art.
The technical scheme for realizing the purpose of the invention is as follows: the rare earth nano fluorescence detection card comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, and a sample pad, a coating film and absorbent paper which are sequentially overlapped and stuck on the bottom lining along the length direction of the bottom lining; the sample pad is sprayed with a microsphere line; the microsphere line is a cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and a rabbit IgG antibody marked by rare earth nano fluorescent microspheres; the coating film comprises a nitrocellulose membrane, and a first detection line, a second detection line, a third detection line, a fourth detection line, a fifth detection line and a quality control line which are sequentially arranged on the nitrocellulose membrane in parallel at intervals along the length direction; the first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line are respectively coated with cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 and goat anti-rabbit antibody, wherein the first detection line is close to the sample pad, and the quality control line is far away from the sample pad.
The content of the cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and the content of the rabbit IgG antibody marked by the rare earth nano fluorescent microspheres sprayed on the sample pad are both 50-200 mug antibody/200 mul fluorescent microspheres.
The rare earth nanometer fluorescent microsphere is prepared by coating neodymium-doped gadolinium lutetium fluoride with yttrium sodium fluoride in a core-shell structure, the particle size is 40 nm-60 nm, and the rare earth nanometer fluorescent microsphere comprises the following components:
NaGdxLu1-y-xF4:yNd@NaYF4，
wherein NaGdxLu1-xF4 is used as a matrix, and the doping ions are neodymium (Nd)3+) (ii) a The colon represents neodymium doping; x and y are the rare earth ion doping mole percentage, the range of x is 20-90%, and the range of y is 1-10%;
the rare earth nano fluorescent microspheres are stable under a ground state, and emit fluorescence with the wavelength range of 1000-1100 nm under the action of an excitation light source of 800-1000 nm.
And the spacing distance between the first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line on the nitrocellulose membrane is 2-4 mm.
The coating concentration of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody on the nitrocellulose membrane is 0.1-2 mg/ml, the coating liquid amount is 0.5-1.5 microliter/cm of the membrane, and the coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg/ml, and the coating liquid amount is 0.5-1.5 microliter/cm of the membrane.
The preparation method of the detection test strip comprises the following steps:
step I, synthesizing NaGdxLu1-y-xF4: yNd (neodymium-doped gadolinium lutetium fluoride): adding a mixture of 3-6 by volume: 7-14 parts of oleic acid and 1-octadecene, and then adding x parts of nitrate or acetate or chloride of Gd, y parts of nitrate or acetate or chloride of neodymium, and 1-x-y parts of nitrate or acetate or chloride of Lu according to molar ratio, wherein x and y are the rare earth ion doping mole percentage, the range of x is 20-70%, and the range of y is 0-30%; mixing and stirring at room temperature, vacuumizing, heating to 100-120 ℃, reacting for 20-30 minutes, heating to 150-160 ℃, and reacting for 10-15 minutes to obtain a transparent solution; naturally cooling to 40-50 ℃, releasing vacuum, and adding substances in a ratio of 1-2: 1.6-3.4 of mixed solution of NaOH and ammonium fluoride methanol, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-300 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane ethanol mixed solution, and dispersing in cyclohexane;
step two, preparing NaGdxLu1-y-xF4: yNd @ NaYF4 (core-shell structure neodymium-doped gadolinium lutetium fluoride-coated yttrium sodium fluoride nano microspheres): adding a mixture of 3-6 by volume: adding 7-14 oleic acid and 1-octadecene into yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of NaOH and ammonium fluoride methanol, and mixing with oleic acid in a volume ratio of 3-6: 2-4 parts of NaGdxLu1-y-xF4: yNd nanometer probe cyclohexane solution, mixing and stirring, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-290 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane ethanol mixed solution, and dispersing in cyclohexane; the weight ratio of the yttrium acetate, NaOH and ammonium fluoride is 0.2-0.4: 0.5-1: 0.8 to 1.6; transferring the probe to a water phase by using an acid washing method, and modifying carboxyl on the surface of the probe to obtain the water-soluble NaGdxLu1-y-xF4: yNd @ NaYF4 rare earth nano fluorescent microsphere with good dispersibility;
step three, activation of the rare earth nano fluorescent microspheres: after the fluorescent microspheres are subjected to ultrasonic treatment for 1-2 min, centrifuging the fluorescent microspheres at a high speed of 12000-14000 rpm for 5-15 min, washing precipitates with 10-100 mM MES solution with the pH of 5.0-7.0, and performing ultrasonic treatment for 2-3 min; adding 20-100 mg/ml carbodiimide, uniformly mixing for 5-10 min, adding 20-100 mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, then centrifuging at 12000-14000 rpm for 5-15 min at high speed, and washing the precipitate with MES solution with pH of 5.0-7.0;
preparing a rare earth nano fluorescent microsphere labeled cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody: carrying out ultrasonic treatment on the activated fluorescent microspheres for 1-2 min, adding a cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody according to 50-200 microgram/200 microliter, uniformly mixing for 1-3 hours, carrying out high-speed centrifugation for 5-15 min at 12000-14000 rpm after sealing for 0.5-1 hour by using 10-50 mM containing 0.5% BSA, and pH7.5-8.5 Tris-HCl sealing liquid, washing and resuspending by using 10-50 mM containing 1% NaCl, 0.5% BSA, and 0.1% Tween-20, and pH7.5-8.5 Tris-HCl preserving liquid, and storing in a dark place at 4 ℃;
preparing a sample pad: uniformly spraying two lines in parallel on one side, close to the sample pad, of the bottom lining by using a sample pad treatment solution, wherein the usage amount is 2-4 mu l of liquid/cm of the sample pad, uniformly spraying one line, which is diluted by 8-30 times, of cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and rabbit IgG antibody marked by rare earth nano fluorescent microspheres, on one side, close to the coating film, of the sample pad by using a microsphere dissociation solution, wherein the usage amount is 2-4 mu l of liquid/cm of the sample pad, placing the sample pad in an oven, and drying the sample pad overnight at 37 ℃; the sample pad treatment solution is Tris-HCl with 0.5% NaCl, 0.5% S17, 0.1% BSA, 20mM of 1mg/ml anti-RBC antibody, pH 8.0; the microsphere dissociation solution is 20mM Tris-HCl buffer solution containing 0.5% BSA and 25% sucrose;
step sixthly, preparing a coating film: respectively adjusting the concentration of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody and the goat anti-rabbit IgG antibody to 0.5-2 mg/ml by using a coating buffer solution, wherein the amount of the coating solution is 0.5-1.5 microliter per cm of membrane, the coating solution is respectively used as a first detection line, a second detection line, a third detection line, a fourth detection line, a fifth detection line and a quality control line which are parallelly drawn on a nitrocellulose membrane for coating in a mode of 2-4 mm interval, and then the nitrocellulose membrane is placed in an oven and dried at 45 ℃ overnight;
and step seventhly, adhering a sample pad, a coating film and absorbent paper to the bottom lining along the length direction in sequence in a mutually overlapping manner to obtain a test paper board, and cutting to obtain the test paper strip.
The clamping shell comprises a plastic lower shell and a plastic upper shell which are connected in a buckling manner; the detection test strip is fixed on the plastic lower shell, and the surface of the detection test strip is tightly pressed through the plastic upper shell; and the plastic upper shell is provided with a sample adding hole and an observation window respectively at the positions corresponding to the sample pad and the coating film.
The invention also provides a heart-lung quintuplet detection kit, which comprises a rare earth nano fluorescence detection card and an ID card containing a standard curve; the rare earth nanometer fluorescence detection card adopts the rare earth nanometer fluorescence detection card; the ID card containing the standard curve is used for detecting a calibrator of gradient concentration through a detection test strip, the concentration of the calibrator is used as an abscissa, a fluorescence signal ratio is used as an ordinate, the calibrator is drawn into the standard curve, and corresponding two-dimensional code information is written in and generated and stored in the ID card to obtain the calibrator. The dry-type fluorescence immunoassay instrument can read the corresponding two-dimensional code information on the rare earth nanometer fluorescence detection card and measure the corresponding concentration.
The method for quantitatively detecting the cTnI/CKMB/Myo/D-Dimer/NT-proBNP by using the cardiopulmonary quintuplet detection kit comprises the following steps:
step one, placing the detection kit and a sample at room temperature, and using the detection kit and the sample after the temperature is restored to room temperature;
step two, starting a rare earth nano fluorescence immunoassay analyzer, preheating for 5min, and then inserting a corresponding ID card;
accurately sucking 100 mu L of blood to be detected and adding the blood to the sample adding hole of the detection card;
and step four, inserting the detection card into the detection slot, and detecting, reading and printing the detection result after 10 min.
The detection principle of the heart-lung quintuplet detection kit is a double-antibody sandwich method, and the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in human serum, plasma and whole blood samples is detected. Dropwise adding a blood sample diluent containing cTnI/CKMB/Myo/D-Dimer/NT-proBNP into a sample adding area, carrying out capillary chromatography to a sample pad, combining with cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 marked by time-resolved fluorescent microspheres to form a microsphere antibody-antigen complex, carrying out chromatography to a detection area on a nitrocellulose membrane, capturing the reaction complex by a cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 antibody coated by a detection line, continuously carrying out forward chromatography on the reaction complex, and combining rabbit IgG with a goat-anti-rabbit fixed on a quality control line. And scanning and detecting the detection area by using a light source, wherein the rare earth nano fluorescent microspheres on the detection area and the quality control line emit fluorescence, the strength of a signal is in direct proportion to the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in a sample, and the autofluorescence of an object in the fluorescence range is weak. By delaying the measurement time, the specific fluorescence of the rare earth element is measured after the naturally occurring short-lived fluorescence in the sample matrix is completely decayed, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the substance to be detected in the sample can be analyzed through the strength and the ratio of the fluorescence intensity of the detection line and the fluorescence intensity of the quality control line.
By adopting the technical scheme, the invention has the following beneficial effects: the cardiopulmonary quintuplet detection kit can accurately and quantitatively detect the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in human blood, can avoid the fluorescence interference of a sample by utilizing a rare earth nanometer fluorescent microsphere detection technology, has the advantages of low background, long luminous life, strong fluorescent signal, high signal to noise ratio and the like because the fluorescent microsphere is a rare earth fluoride near-infrared two-region luminescent nano material, is characterized by connecting the microsphere and an antibody by a covalent bond, stable labeled product, wide detection range, high sensitivity, high accuracy, quick, simple and convenient detection and the like, and can help a doctor to accurately diagnose the cardiopulmonary function in early stage.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a schematic structural diagram of a rare earth nano fluorescence detection card of the present invention.
FIG. 2 is a schematic structural diagram of the test strip of the present invention.
FIG. 3 is a standard curve chart of the cardiopulmonary quintuplet detection kit of the present invention, plotted with the cTnI quality control concentration and the sample signal T/C average value.
FIG. 4 is a standard curve chart for plotting CKMB quality control substance concentration and sample signal T/C average value of the heart-lung quintuplet detection kit.
FIG. 5 is a standard curve chart for plotting Myo quality control concentration and sample signal T/C average value of the cardiopulmonary five-link detection kit.
FIG. 6 is a standard curve chart of the cardiopulmonary quintuplet detection kit D-Dimer quality control concentration and sample signal T/C average value.
FIG. 7 is a standard curve chart of the quality control substance concentration of NT-proBNP and the average value of sample signal T/C drawn by the cardiopulmonary quintuplet detection kit.
Fig. 8 is a comparison graph of the detection results of the cardiopulmonary quintuplet detection kit and the cTnI detection kit by the beckmann chemiluminescence method on the same sample.
FIG. 9 is a comparison graph of the detection results of the cardiopulmonary quintuplet detection kit and the Myo detection kit of the Beckmann chemiluminescence method on the same sample.
Fig. 10 is a comparison graph of the detection results of the cardiopulmonary quintuplet detection kit and the CKMB detection kit of the present invention on the same sample by the beckmann chemiluminescence method.
FIG. 11 is a comparison graph of the detection results of the cardiopulmonary pentahapto detection kit of the present invention and the Siemens immunoturbidimetry detection D-dimer detection kit on the same sample.
FIG. 12 is a comparison graph of the detection results of the cardiopulmonary quintuplet assay kit and the kit for detecting NT-proBNP by the Roche electrochemiluminescence method of the present invention on the same sample.
The reference numbers in the figures are:
the device comprises a clamping shell 1, a sample adding hole 11 and an observation window 12;
the test strip 2, a bottom liner 21, a sample pad 22, a coating film 23 and absorbent paper 24.
Detailed Description
(example 1)
Referring to fig. 1 and fig. 2, the cardiopulmonary quintuplet assay kit of the present embodiment includes a rare earth nano fluorescence detection card and an ID card containing a standard curve.
The rare earth nanometer fluorescence detection card comprises a card shell 1 and a detection test strip 2 arranged in the card shell 1. The detection test strip 2 comprises a bottom liner 21, a sample pad 22, a coating film 23 and absorbent paper 24, wherein the sample pad 22, the coating film 23 and the absorbent paper 24 are sequentially overlapped and stuck on the bottom liner along the length direction of the bottom liner; the sample pad 22 is a sample application region for sucking a blood test sample to be tested. The sample pad 22 is sprayed with microsphere lines; the microsphere line is cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and rabbit IgG antibody (the content is 100 mug antibody/200 mug fluorescent microsphere). The rare earth nanometer fluorescent microsphere is neodymium-doped gadolinium lutetium fluoride coated sodium yttrium fluoride with a core-shell structure, the particle size is 60nm, and the rare earth nanometer fluorescent microsphere comprises the following components: NaGdxLu1-y-xF4: yNd @ NaYF4, wherein NaGdxLu1-xF4 is used as a matrix, and doping ions are neodymium (Nd 3 +); the colon represents neodymium doping; x and y are the doping mole percentage of rare earth ions, the range of x is 20-90%, and the range of y is 1-10%; the rare earth nano fluorescent microspheres are stable under a ground state, and emit fluorescence with the wavelength range of 1064nm under the action of an excitation light source of 808 nm.
The coating film 23 comprises a nitrocellulose membrane, and a first detection line, a second detection line, a third detection line, a fourth detection line, a fifth detection line and a quality control line which are sequentially arranged on the nitrocellulose membrane in parallel at intervals along the length direction. The spacing distance between the first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line is 2-4 mm. The first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line are respectively coated with cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 and goat anti-rabbit antibody, wherein the first detection line is close to the sample pad 22, and the quality control line is far away from the sample pad 22. The coating concentration of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody is 1mg/ml, the dosage is 1 mul of coating liquid volume/cm membrane, and the coating concentration of the goat anti-rabbit IgG antibody is 1mg/ml, and the dosage is 1 mul of coating liquid volume/cm membrane.
The preparation method of the test strip 2 comprises the following steps:
step I, synthesizing NaGdxLu1-y-xF4: yNd (neodymium-doped gadolinium lutetium fluoride): adding 4.5 mL of oleic acid and 12.5 mL of 1-octadecene into a 100mL three-neck round-bottom flask, adding 1.6 mmol of gadolinium acetate, 1.6 mmol of lutetium acetate and 0.02 mmol of neodymium acetate according to a molar ratio, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of 1 mmol of NaOH and 1.6 mmol of ammonium fluoride methanol, and reacting for 30 min; heating to 100 deg.C, extracting air, ventilating for 3 times, introducing nitrogen, heating to 300 deg.C, reacting for 1.5 hr, centrifuging at 6000 rpm, washing with cyclohexane-ethanol mixture for 3 times, and dispersing in cyclohexane. (ii) a
Step two, preparing NaGdxLu1-y-xF4: yNd @ NaYF4 (core-shell structure neodymium-doped gadolinium lutetium fluoride-coated yttrium sodium fluoride nano microspheres): adding 3 mL of oleic acid and 7 mL of 1-octadecene into a 100mL three-neck round-bottom flask, adding 0.4 mmol of yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of 1 mmol of NaOH and 1.6 mmol of ammonium fluoride methanol and 4 mL of NaGdxLu1-y-xF4: yNd nano probe cyclohexane solution, mixing and stirring, and reacting for 30 min; heating to 100 ℃, extracting air and ventilating for 3 times, introducing nitrogen, heating to 290 ℃, reacting for 1 hour, centrifuging at 6000 rpm, washing for 3 times by using cyclohexane-ethanol mixed liquor, and dispersing in cyclohexane; transferring the probe to a water phase by using an acid washing method, and modifying carboxyl on the surface of the probe to obtain the water-soluble NaGdxLu1-y-xF4: yNd @ NaYF4 rare earth nano fluorescent microsphere with good dispersibility. The mole percent of x in the NaGdxLu1-y-xF4: yNd nano probe is preferably 40%, and the mole percent of y in the nano probe is preferably 5%. The size of the material is about 60nm, the appearance is uniform, and the luminescence performance is good. The excitation wavelength of the nano probe is 808nm, and the emission wavelength is 1064 nm.
Step three, activation of the rare earth nano fluorescent microspheres: after the fluorescent microspheres are subjected to ultrasonic treatment for 2min, 200 microliters of fluorescent microspheres are centrifuged at 14000rpm for 15min, and precipitates are washed to 1ml by MES solution with the concentration of 100mM and the pH value of 6.0 and subjected to ultrasonic treatment for 2 min; adding 50 mul of 100mg/ml carbodiimide, uniformly mixing for 5min, adding 100 mul of 100mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 15min, then carrying out high-speed centrifugation at 14000rpm for 15min, and washing the precipitate to 1ml by using MES solution with the pH value of 6.0.
Preparing a rare earth nano fluorescent microsphere labeled cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody: and (3) after the fluorescent microspheres activated in the step (III) are subjected to ultrasonic treatment for 2min, adding the cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and the rabbit IgG antibody according to 100 microgram/200 microliter, uniformly mixing for 2 hours, sealing for 1 hour by using 50mM containing 0.5% BSA and pH8.0 Tris-HCl sealing solution, then centrifuging at 14000rpm for 15min at high speed, washing twice by using buffer solution in 50mM containing 1% (w/w) NaCl, 0.5% (w/w) BSA, 0.1% (w/w) Tween-20 and pH8.0 Tris-HCl preservation solution, and carrying out ultrasonic treatment to resuspend 200 microliter and preserving in a dark place at 4 ℃.
Preparing a sample pad 22: two lines are evenly sprayed in parallel on one side, close to the sample pad, of the bottom lining 21 by using a sample pad treatment solution (containing 0.5% NaCl, 0.5% S17, 0.1% BSA, 20mM of 1mg/ml anti-RBC antibody and Tris-HCl with pH 8.0) in an amount of 4 mu l/cm, and a cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody marked by rare earth nano fluorescent microspheres and a rabbit IgG antibody are evenly sprayed on one line by using a microsphere diluent (containing 0.5% (w/w) BSA and 25% (w/w) sucrose and 20mM Tris-Hcl buffer solution in an amount of 4 mu l/cm by using a sample pad treatment solution. The mixture was placed in an oven and dried overnight at 37 ℃.
Step sixthly, preparing a coating film 23: respectively adjusting the concentration of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody and the goat anti-rabbit IgG antibody to 1mg/ml by using a coating buffer solution (20 mM Tris-HCl buffer solution with pH8.0 and containing 2.5% (w/w) sucrose), respectively taking 1 mu l of coating solution/cm membrane as a detection line and a quality control line, respectively, parallelly scratching the membrane on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 4mM, airing the membrane in an oven with the humidity of less than 30% and the temperature of 37 ℃ for 10 hours, and sealing the membrane for later use
Seventhly, the sample pad 22 (30 mm 300mm in size, made of glass fiber cotton), the coating film 23 (25 mm 300mm in size, made of cellulose nitrate) and the absorbent paper 24 (28 mm 300mm in size) are sequentially adhered to the bottom liner 21 (80 mm 300mm in size) in an overlapping manner to obtain a test paper board, and the test paper board is cut into test strips 2 with the width of 4mm according to requirements.
The clamping shell 1 comprises a plastic lower shell and a plastic upper shell which are connected in a buckling manner; the detection test strip 2 is fixed on the plastic lower shell, and the surface of the detection test strip 2 is compressed tightly through the plastic upper shell. The plastic upper shell is provided with a sample adding hole 11 and an observation window 12 respectively corresponding to the positions of the sample pad 22 and the coating film 23.
The ID card containing the standard curve measures a calibrator of gradient concentration through the detection test strip 2, the concentration of the calibrator is used as an abscissa, the ratio of the fluorescence signals is used as an ordinate, the calibrator is drawn into the standard curve, and corresponding two-dimensional code information is written and generated and stored in the ID card to obtain the calibrator. The dry-type fluorescence immunoassay instrument can read the corresponding two-dimensional code information on the rare earth nanometer fluorescence detection card and measure the corresponding concentration. The standard curves of the ID cards containing the standard curves in the same batch of kits are the same.
The method for quantitatively detecting the cTnI/CKMB/Myo/D-Dimer/NT-proBNP by using the cardiopulmonary quintuplet detection kit comprises the following steps:
placing a detection kit and a sample at room temperature, and using the detection kit and the sample after the detection kit and the sample are recovered to the room temperature;
step two, starting the rare earth nanometer fluorescence immunoassay analyzer, preheating for 5min, and then inserting a corresponding ID card;
step three, accurately sucking 100 mu L of blood to be detected and adding the blood to be detected into the sample adding hole of the detection card;
and step four, inserting the detection card into the detection slot, and detecting, reading and printing the detection result after 10 min.
The detection principle of the cardiopulmonary quintuplet detection kit is a double-antibody sandwich method, and the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in human serum, plasma and whole blood samples is detected. Dropwise adding blood sample diluent containing cTnI/CKMB/Myo/D-Dimer/NT-proBNP into a sample adding area, carrying out capillary chromatography to a sample pad, combining with cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 marked by time-resolved fluorescent microspheres to form a microsphere antibody-antigen complex, carrying out chromatography to a detection area on a nitrocellulose membrane, capturing the reaction complex by a cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 antibody coated by a detection line, continuously carrying out forward chromatography on redundant fluorescent microsphere markers, and combining rabbit IgG with goat anti-rabbit antibodies fixed on a quality control line. And (2) scanning and detecting the detection area by using a light source (808 nm), wherein the rare earth nano fluorescent microspheres on the detection line and the quality control line emit fluorescence (1064 nm), the strength of a signal is in direct proportion to the content of cTnI/CKMB/Myo/D-Dimer/NT-proBNP in a sample, and the autofluorescence of an object in the fluorescence range is weak. By using the delayed measurement time, after the naturally occurring short-life fluorescence (1-10 ns) in the sample matrix is completely decayed, the specific fluorescence of the rare earth elements is measured, so that the interference of the specific background fluorescence can be completely eliminated. The concentration of the substance to be detected in the sample can be analyzed through the strength and the ratio of the fluorescence intensity of the detection line and the fluorescence intensity of the quality control line.
The standard curve of the cardiopulmonary quintuplet detection kit of the present embodiment is drawn by the following process: adding cTnI/CKMB/Myo/D-Dimer/NT-proBNP antigen quality control products (three repeats are arranged at each concentration and are obtained by diluting cTnI/CKMB/Myo/D-Dimer/NT-proBNP antigens with 20% calf serum) with different concentrations into the prepared rare earth nano fluorescent test paper card, performing sample addition chromatography for 10min, and reading C, T line fluorescent signals and C/T values by a rare earth nano fluorescent immunoassay analyzer with exciting light (808 nm)/emitting light (1064 nm), wherein the experimental results and analysis are shown in Table 1:
and (3) drawing a standard curve by using the concentration of the cTnI/CKMB/Myo/D-Dimer/NT-proBNP quality control product and the T/C average value of the sample signal, wherein the curve data are shown in Table 1, and the standard curve is shown in figures 3 to 7. Wherein the R values of the cTnI/CKMB/Myo/D-Dimer/NT-proBNP are 0.9979, 0.9939, 0.9933, 0.9966 and 0.9958 respectively, and the concentrations of the cTnI/CKMB/Myo/D-Dimer/NT-proBNP contained in the sample are quantitatively determined through the marked lines.
The performance test of the rare earth nanometer fluorescent test paper card of the heart and lung quintuplet detection kit comprises the following steps:
(1) minimum detection limit: the measurement is repeated for 20 times by using a zero-value sample, the mean M and the standard deviation SD of the results of 20 times are calculated, the detection limit (M +2 SD) of the reporting method is added by two times of the standard deviation by using a blank mean, and the cTnI/CKMB/Myo/D-Dimer/NT-proBNP results are respectively 0.07ng/mL, 0.67ng/mL, 1.8ng/mL, 0.07mg/L and 9.21pg/mL, and respectively meet the sensitivity standards of 0.1ng/mL, 1ng/mL, 2.5ng/mL, 0.1mg/L and 10 pg/mL.
(2) Linear range: respectively taking seven concentration values among 0.1-30 ng/mL of cTnI, 1-60 ng/mL of CKMB, 5-400 ng/mL of Myo and 0.1-10 mg/L, NT of D-Dimer, 15-15000 pg/mL of proBNP, repeatedly measuring each concentration for three times, and carrying out linear analysis on the average value of the measured concentration at the theoretical concentration to obtain a cTnI linear equation y = 0.9774x +0.2923 and r =0.9982, a CKMB linear equation y =0.9802x +0.2478 and r =0.9981, a Myo linear equation y =0.9925x +2.7953 and r = 0.9984; D-Dimer linear equation y =1.0059x +0.0255, r = 0.9984; NT-proBNP linear equation y =0.9558x +1.3976, r = 0.9993; the correlation of the heart-lung quintuplet detection kit disclosed by the invention in a linear range is very good.
(3) Precision: taking three batches of the kit of the embodiment, respectively detecting the CV of the repetitive quality control product in the three batches, and parallelly detecting each batch of the kit by using the repetitive quality control product for 10 times, wherein the CV of cTnI 0.3ng/mL in the three batches is respectively 3.66%, 10.09% and 10.23%, the CV of the cTnI between the three batches is 8.95%, the CV of the cTnI 10.3 ng/mL in the three batches is respectively 7.05%, 7.59% and 8.98%, and the CV of the cTnI between the three batches is 7.69%; the CV of CKMB in three batches of 5ng/mL is respectively 8.31%, 7.81% and 5.82%, the CV between batches is 7.22%, the CV of CKMB in three batches of 30ng/mL is respectively 8.38%, 8.74% and 9.35%, and the CV between batches is 8.55%; the CV in Myo 50ng/mL three batches is respectively 8.23%, 7.85% and 7.75%, the CV between batches is 7.90%, the CV in 300 ng/mL three batches is respectively 6.86%, 7.27% and 7.25%, and the CV between batches is 7.08%; the CV of the D-Dimer in three batches of 0.6 mg/L is respectively 5.03%, 6.66% and 9.03%, the CV between the batches is 6.86%, the CV of the D-Dimer in three batches of 5mg/L is respectively 8.41%, 7.05% and 8.09%, and the CV between the batches is 7.62%; NT-proBNP 300pg/mL three batches of internal CVs were 7.46%, 8.78%, 6.50%, respectively, the batch-to-batch CV was 7.46%, 5000pg/mL three batches of internal CVs were 5.85%, 9.52%, 8.98%, respectively, the batch-to-batch CV was 8.10%; are all within 10%.
(4) Accuracy: selecting the quality control material of the basic sample as a detection sample, dividing the detection sample into 3 parts with the same volume, respectively adding accuracy quality control materials with different concentrations into 2 parts of the sample, preparing 2 recovery samples with different addition concentrations, and calculating the concentration of the added object to be detected. The same amount of the analyte-free solution was added to the other sample to prepare a base sample. The samples were analyzed for 3 replicates and the mean value was calculated. Calculated recovery = (assay sample concentration-base sample concentration)/added concentration × 100%. The recovery rate of 20ng/mL of the cTnI recovered sample is 105.60%, the recovery rate of 5ng/mL is 96.07%, and the average recovery rate is 100.83%; the recovery rate of the CKMB recovery sample is 103.33% at 50ng/mL, 89.47% at 20ng/mL and 96.40% on average; the recovery rate of the Myo recovery sample 400ng/mL is 98.71%, the recovery rate of the Myo recovery sample 150ng/mL is 101.47%, and the average recovery rate is 100.09%; the recovery rate of 8mg/L of the D-Dimer recovery sample is 92.58 percent, the recovery rate of 2.5mg/L is 110.00 percent, and the average recovery rate is 101.29 percent; the recovery rate of 10000pg/mL of the recovered sample of NT-proBNP is 103.02%, the recovery rate of 800pg/mL is 98.96%, and the average recovery rate is 100.99%. The deviation is within 10%.
The clinical samples of the cardiopulmonary quintuplet assay kit of the present embodiment are tested as follows:
100 parts of cTnI/CKMB/Myo/D-Dimer/NT-proBNP blood samples detected by hospitals are collected, and the kit is used for detection comparison with a kit for detecting NT-proBNP by a Roche electrochemical luminescence method, a kit for detecting cTnI, Myo and CKMB by a Beckmann chemiluminescence method and a kit for detecting D-Dimer by a Siemens immunoturbidimetry method. In the kit, 80 mul of blood sample is added into a sample adding hole of a detection card, the concentration is read by a rare earth nanometer fluorescence immunoassay analyzer after chromatography for 10min, and the concentration of the same sample is detected by a Roche electrochemical luminescence method NT-proBNP detection kit, a Beckmann chemiluminescence method cTnI detection kit, Myo detection kit and CKMB detection kit and a Siemens immunoturbidimetry detection D-dimer kit. The detection results are subjected to linear analysis, as shown in fig. 8 to fig. 12, the correlation is good, cTnI r =0.9861, Myo r =0.9833, CKMB r =0.9869, NT-proBNP r =0.9864, D-Dimer r =0.9850, P >0.05, and the average relative deviation is less than 10%, so that the detection results meet the requirements of clinical analysis and are suitable for clinical detection.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The rare earth nanometer fluorescence detection card is characterized in that: comprises a card shell (1) and a detection test strip (2) arranged in the card shell (1); the detection test strip (2) comprises a bottom liner (21), a sample pad (22), a coating film (23) and absorbent paper (24), wherein the sample pad is sequentially overlapped and stuck on the bottom liner along the length direction of the bottom liner; the sample pad (22) is sprayed with microsphere lines; the microsphere line is a cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and a rabbit IgG antibody marked by rare earth nano fluorescent microspheres; the coating film (23) comprises a nitrocellulose membrane, and a first detection line, a second detection line, a third detection line, a fourth detection line, a fifth detection line and a quality control line which are sequentially arranged on the nitrocellulose membrane in parallel at intervals along the length direction; the first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line are respectively coated with cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 and goat anti-rabbit antibody, wherein the first detection line is close to the sample pad (22), and the quality control line is far away from the sample pad (22);
the rare earth nanometer fluorescent microsphere is prepared by coating neodymium-doped gadolinium lutetium fluoride with yttrium sodium fluoride in a core-shell structure, the particle size is 40 nm-60 nm, and the rare earth nanometer fluorescent microsphere comprises the following components:
NaGdxLu1-y-xF4:yNd@NaYF4，
wherein, NaGdxLu1-xF4 is used as a matrix, and the doping ion is Nd3+(ii) a The colon represents neodymium doping; x and y are the doping mole percentage of rare earth ions, the range of x is 20-90%, and the range of y is 1-10%;
the rare earth nano fluorescent microspheres are stable under a ground state, and emit fluorescence with the wavelength range of 1000-1100 nm under the action of an excitation light source of 800-1000 nm;
the preparation method of NaGdxLu1-y-xF4: yNd @ NaYF4 comprises the following steps:
step I, synthesizing NaGdxLu1-y-xF4: yNd: adding a mixture of 3-6 by volume: 7-14 parts of oleic acid and 1-octadecene, and then adding x parts of nitrate or acetate or chloride of Gd, y parts of nitrate or acetate or chloride of neodymium, and 1-x-y parts of nitrate or acetate or chloride of Lu according to molar ratio, wherein x and y are the rare earth ion doping mole percentage, the range of x is 20-70%, and the range of y is 0-30%; mixing and stirring at room temperature, vacuumizing, heating to 100-120 ℃, reacting for 20-30 minutes, heating to 150-160 ℃, and reacting for 10-15 minutes to obtain a transparent solution; naturally cooling to 40-50 ℃, releasing vacuum, and adding the materials in a ratio of 1-2: 1.6-3.4 of mixed solution of NaOH and ammonium fluoride methanol, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-300 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane ethanol mixed solution, and dispersing in cyclohexane;
step two, preparing NaGdxLu1-y-xF4: yNd @ NaYF 4: adding a mixture of 3-6 by volume: adding 7-14 parts of oleic acid and 1-octadecene into yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of NaOH and ammonium fluoride methanol, and mixing with oleic acid in a volume ratio of 3-6: 2-4 of NaGdxLu1-y-xF4: yNd nano probe cyclohexane solution, mixing and stirring, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-290 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane ethanol mixed solution, and dispersing in cyclohexane; the weight ratio of the yttrium acetate, NaOH and ammonium fluoride is 0.2-0.4: 0.5-1: 0.8 to 1.6; transferring the probe to a water phase by using an acid washing method, and modifying carboxyl on the surface of the probe to obtain the water-soluble NaGdxLu1-y-xF4: yNd @ NaYF4 rare earth nano fluorescent microsphere with good dispersibility.
2. The rare earth nanofluorescent detection card according to claim 1, characterized in that: the content of the cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and the content of the rabbit IgG antibody marked by the rare earth nano fluorescent microspheres on the sample pad (22) are both 50-200 mug antibody/200 mug fluorescent microspheres.
3. The rare earth nanofluorescent detection card according to claim 1, characterized in that: and the spacing distance between the first detection line, the second detection line, the third detection line, the fourth detection line, the fifth detection line and the quality control line on the nitrocellulose membrane is 2-4 mm.
4. The rare earth nanofluorescent detection card according to claim 3, wherein: the coating concentration of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody on the nitrocellulose membrane is 0.1-2 mg/ml, the coating amount is 0.5-1.5 microliter coating liquid amount/cm membrane, and the coating concentration of the goat anti-rabbit IgG antibody is 0.5-2 mg/ml, and the coating amount is 0.5-1.5 microliter coating liquid amount/cm membrane.
5. The rare earth nanofluorescent detection card according to claim 4, characterized in that: the preparation method of the detection test strip (2) comprises the following steps:
step I, synthesizing NaGdxLu1-y-xF4: yNd: adding a mixture of 3-6 by volume: 7-14 parts of oleic acid and 1-octadecene, and then adding x parts of nitrate or acetate or chloride of Gd, y parts of nitrate or acetate or chloride of neodymium, and 1-x-y parts of nitrate or acetate or chloride of Lu according to molar ratio, wherein x and y are the rare earth ion doping mole percentage, the range of x is 20-70%, and the range of y is 0-30%; mixing and stirring at room temperature, vacuumizing, heating to 100-120 ℃, reacting for 20-30 minutes, heating to 150-160 ℃, and reacting for 10-15 minutes to obtain a transparent solution; naturally cooling to 40-50 ℃, releasing vacuum, and adding substances in a ratio of 1-2: 1.6-3.4 of mixed solution of NaOH and ammonium fluoride methanol, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-300 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane-ethanol mixed solution, and dispersing in cyclohexane;
step two, preparing NaGdxLu1-y-xF4: yNd @ NaYF 4: adding a mixture, by volume, of 3-6: adding 7-14 parts of oleic acid and 1-octadecene into yttrium acetate, mixing and stirring at room temperature, vacuumizing, heating to 120 ℃, reacting for 20 minutes, heating to 160 ℃, and reacting for 10 minutes to obtain a transparent solution; naturally cooling to 50 ℃, releasing vacuum, adding a mixed solution of NaOH and ammonium fluoride methanol, and mixing with oleic acid in a volume ratio of 3-6: 2-4 of NaGdxLu1-y-xF4: yNd nano probe cyclohexane solution, mixing and stirring, and reacting for 20-30 min; heating to 90-100 ℃, exhausting air for 3-4 times, introducing nitrogen, heating to 280-290 ℃, reacting for 1-2 hours, centrifuging, washing for 3-4 times by using a cyclohexane ethanol mixed solution, and dispersing in cyclohexane; the weight ratio of the yttrium acetate, NaOH and ammonium fluoride is 0.2-0.4: 0.5-1: 0.8 to 1.6; transferring the probe to a water phase by using an acid washing method, and modifying carboxyl on the surface of the probe to obtain the water-soluble NaGdxLu1-y-xF4: yNd @ NaYF4 rare earth nano fluorescent microsphere with good dispersibility;
activating the rare earth nanometer fluorescent microspheres: after the fluorescent microspheres are subjected to ultrasonic treatment for 1-2 min, centrifuging the fluorescent microspheres at a high speed of 12000-14000 rpm for 5-15 min, washing precipitates with 10-100 mM MES solution with the pH of 5.0-7.0, and performing ultrasonic treatment for 2-3 min; adding 20-100 mg/ml carbodiimide, uniformly mixing for 5-10 min, adding 20-100 mg/ml N-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, then centrifuging at 12000-14000 rpm for 5-15 min at a high speed, and washing the precipitate with an MES solution with the pH of 5.0-7.0;
preparing a rare earth nano fluorescent microsphere labeled cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody: carrying out ultrasonic treatment on the activated fluorescent microspheres for 1-2 min, adding a cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody according to 50-200 microgram/200 microliter, uniformly mixing for 1-3 hours, carrying out high-speed centrifugation for 5-15 min at 12000-14000 rpm after sealing for 0.5-1 hour by using 10-50 mM containing 0.5% BSA, and pH7.5-8.5 Tris-HCl sealing liquid, washing and resuspending by using 10-50 mM containing 1% NaCl, 0.5% BSA, and 0.1% Tween-20, and pH7.5-8.5 Tris-HCl preserving liquid, and storing in a dark place at 4 ℃;
preparing a sample pad (22): uniformly spraying two lines in parallel on one side, close to the sample pad, of a bottom lining (21) by using a sample pad treatment solution, wherein the amount of the two lines is 2-4 mu l of solution/cm of the sample pad, uniformly spraying one line, which is diluted by 8-30 times, of cTnI 1/CKMB 1/Myo 1/D-Dimer 1/NT-proBNP 1 monoclonal antibody and rabbit IgG antibody marked by rare earth nano fluorescent microspheres, on one side, close to a coating film (23), of the sample pad (22), wherein the amount of the two lines is 2-4 mu l of solution/cm of the sample pad, placing the sample pad in an oven, and drying the sample pad overnight at 37 ℃; the sample pad treatment solution is 20mM Tris-HCl with pH8.0 containing 0.5% NaCl, 0.5% S17, 0.1% BSA, 1mg/ml anti-RBC antibody; the microsphere dissociation solution is 20mM Tris-HCl buffer solution containing 0.5% BSA and 25% sucrose;
step sixthly, preparing a coating film (23): respectively adjusting the concentrations of the cTnI 2/CKMB 2/Myo 2/D-Dimer 2/NT-proBNP 2 monoclonal antibody and the goat anti-rabbit IgG antibody to 0.5-2 mg/ml by using a coating buffer solution, wherein the amount of the coating solution is 0.5-1.5 microliter per cm of membrane, the coating solution is respectively used as a first detection line, a second detection line, a third detection line, a fourth detection line, a fifth detection line and a quality control line which are parallelly scribed on a nitrocellulose membrane at intervals of 2-4 mm for coating, and then the nitrocellulose membrane is placed in an oven and dried at 45 ℃ overnight;
seventhly, adhering a sample pad (22), a coating film (23) and absorbent paper (24) to the bottom liner (21) along the length direction in sequence in a mutually overlapping manner to obtain a test paper board, and cutting to obtain the test paper strip (2).
6. The rare earth nanofluorescent detection card according to claim 1, characterized in that: the clamping shell (1) comprises a plastic lower shell and a plastic upper shell which are connected in a buckling manner; the detection test strip (2) is fixed on the lower plastic shell, and the surface of the detection test strip (2) is compressed by the upper plastic shell; and the plastic upper shell is provided with a sample adding hole (11) and an observation window (12) at positions corresponding to the sample pad (22) and the coating film (23) respectively.
7. The heart-lung quintuplet detection kit is characterized in that: comprises a rare earth nano fluorescence detection card and an ID card containing a standard curve; the rare earth nano fluorescence detection card adopts the rare earth nano fluorescence detection card of any one of claims 1 to 6; the ID card containing the standard curve measures a calibrator of gradient concentration through the detection test strip (2), the concentration of the calibrator is used as an abscissa, the ratio of the fluorescence signals is used as an ordinate, the calibrator is drawn into the standard curve, and corresponding two-dimensional code information is written and generated and stored in the ID card to obtain the calibrator.
8. The method for quantitatively detecting cTnI/CKMB/Myo/D-Dimer/NT-proBNP by using the cardiopulmonary quintuplet detection kit of claim 7, wherein: the method comprises the following steps:
placing a detection kit and a sample at room temperature, and using the detection kit and the sample after the detection kit and the sample are recovered to the room temperature;
step two, starting the rare earth nanometer fluorescence immunoassay analyzer, preheating for 5min, and then inserting a corresponding ID card;
accurately sucking 100 mu L of blood to be detected and adding the blood to the sample adding hole of the detection card;
and step four, inserting the detection card into the detection slot, and detecting, reading and printing the detection result after 10 min.
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| CN112067824A (en) * | 2020-08-13 | 2020-12-11 | 河南沃迈生物科技有限公司 | BNP detection kit |
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| CN114167052A (en) * | 2021-12-03 | 2022-03-11 | 广州达泰生物工程技术有限公司 | Kit for quantitative detection of NT-proBNP/ST2 by time-resolved fluorescence immunochromatography and application thereof |
| CN114034854A (en) * | 2021-12-03 | 2022-02-11 | 广州达泰生物工程技术有限公司 | Kit for quantitatively detecting cTnT/NT-proBNP/D-dimer and application thereof |
| CN114252597A (en) * | 2021-12-08 | 2022-03-29 | 南京诺唯赞医疗科技有限公司 | Sample pad sealing agent and preparation method and application thereof |
| CN114113636A (en) * | 2021-12-09 | 2022-03-01 | 河北特温特生物科技发展有限公司 | Heart, lung and kidney joint inspection test paper card, kit and preparation method |
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